The present invention relates to a cholesterol sensor capable of performing quantitative determination of cholesterol in a sample with high accuracy in a rapid and simplified manner.
As a system for quantitatively determining a specific component in a sample without requiring dilution or stirring of the sample solution in a rapid and simplified manner, various types of biosensors have heretofore been proposed.
First, a glucose sensor will be described as an example of the biosensors.
As a method for quantitative determination of glucose which utilizes an enzyme electrode, generally known is a system comprising a combination of glucose oxidase and an oxygen electrode or a hydrogen peroxide electrode. The glucose oxidase selectively oxidizes a substrate i.e., .beta.-D-glucose to D-glucono-.delta.-lactone by utilizing oxygen as an electron mediator. By this reaction, oxygen is reduced to hydrogen peroxide. Quantitative determination of glucose is performed by measuring the amount of oxygen consumed by the oxygen electrode in this reaction, or by measuring the amount of hydrogen peroxide produced in this reaction by the hydrogen peroxide electrode of a platinum electrode or the like.
By the above-mentioned method, however, it is impossible to measure glucose concentrations under a condition lacking oxygen. This is why a glucose sensor which does not utilize oxygen but utilizes a metal complex such as potassium ferricyanide, a ferrocene derivative, a quinone derivative or the like, or another organic compound as the electron mediator has been developed. In this type of glucose sensor, reductant of electron mediator produced by enzyme reaction is oxidized by an electrode, and the concentration of glucose is determined by measuring the current required for this oxidation. This mode of measurement has been widely applied to quantitative determination of substrates other than glucose.
As an example of this type of biosensor, a glucose sensor which will be subsequently described is known (Japanese Laid-Open Patent Publication Hei 2-062952).
That is, the disclosed glucose sensor comprises an electrode system of a measuring electrode, a counter electrode and a reference electrode provided on an insulating base plate by means of screen printing or the like, and a reaction layer including a reaction reagent system of a hydrophilic polymer, an oxidoreductase, and an electron mediator, and, if required, a buffer agent is added thereto.
When a sample solution containing a substrate is dropped on the reaction layer, the reaction layer is dissolved and adjusted to a pH value where the highest enzyme activity is obtainable by a buffer action of the buffer agent, the enzyme is allowed to react with the substrate, and the electron mediator is reduced. After completion of the enzyme reaction, the reduced electron mediator is oxidized electrochemically, and the substrate concentration of the sample solution is derived from a current required for the oxidation.
Measurements on various substances are theoretically possible with the biosensors of this type, by using a particular enzyme which has a substrate specificity to the substance to be measured.
When cholesterol oxidase is employed as the oxidoreductase, it is possible to configure a biosensor for measuring cholesterol in serum. Serum cholesterol level used as an index for diagnosis is however a sum of the concentrations of cholesterol and cholesterol ester in serum. Since the cholesterol ester cannot act as the substrate for the oxidation reaction by the cholesterol oxidase, it is required to combine a process of converting the cholesterol ester into cholesterol in order to determine the serum cholesterol level as the index for the diagnosis. This is because the currently employed method is performed on the basis of the following general reaction scheme:
Cholesterol ester+H.sub.2 O.fwdarw.Cholesterol+Fatty acid Cholesterol+Electron mediator (oxidized form).fwdarw.Cholestenone+Electron mediator (reduced form) Electron mediator (reduced form).fwdarw.Electron mediator (oxidized form)
However, if a compound other than oxygen is used as the electron mediator to cause oxidation reaction of cholesterol by the cholesterol oxidase as described above, the rate of secondary reaction between the oxygen and the enzyme becomes faster than the rate of secondary reaction between the electron mediator and the enzyme. This causes a tendency that in the case of dissolution of oxygen in the sample solution, the oxidation current value of the electron mediator measured by the electrode becomes lower than the value estimated to be obtainable by the oxidation reaction of the substrate contained in a sample solution in complete conjugation with the reducing reaction of the electron mediator. The above-mentioned scheme therefore has a problem of inaccurate responses of the sensor particularly to the substrates of low concentrations in the sample solution. Another problem is prolonged reaction time, which poses further problems that an increase in the supported amount of cholesterol oxidase on the sensor in order to avoid time-consuming reaction leads to an increase in manufacturing cost, and that an increase in the supported amount of reagent on the sensor interferes with the fabrication of sensor physically.